Cryopreservation of viable human skin substitutes

ABSTRACT

The present invention relates generally to systems and methods for preparing, storing, shipping and using skin equivalents made by organotypic culture. In particular, the present invention relates to systems and methods for cryopreserving viable skin substitutes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to pending U.S. Provisional PatentApplication No. 61/779,661 filed Mar. 13, 2013, the content of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to systems and methods forcryopreservation of viable human skin substitutes.

BACKGROUND

The emerging field of tissue engineering (TE) is poised to make enormousprogress in the treatment of organ disease and dysfunction in the comingdecade. In 2001, there were 23 cell-based therapeutics approved formarket in the United States (U.S.) and Europe, of which nine were skinsubstitutes or grafts, and 100 more products were in development. (DeBree, Genomics-based Drug Data Report and Regenerative Therapy (1)2:77-96 (2001)). A decade later, engineered tissues have emerged as adiscrete industry sector within the wound care industry and representone of regenerative medicine's most promising cell based therapeuticplatforms. The global wound care market was estimated to be $16.8billion in 2012 and has been growing at a rate of approximately 6%annually (Kalorama Information, April 2012). The bulk of this market iscomprised of traditional sectors that are mature and highly competitive,and include products targeting basic and advanced wound care, woundclosure, and anti-infectives. This has led competitors to increasinglyfocus their attention on developing highly differentiated products inthe more innovative active wound care sector, a sector that representsapproximately 15% of the overall market. While sales of negativepressure wound therapy still predominate, 2010 sales of engineeredtissues and other products within the U.S. biologics market grew to $448million, and are projected to increase to $1.058 billion by 2015, acompound annual growth rate of 18.8% (BioMedGPS-SmarTRAK marketanalysis, 2012).

Although a multitude of revolutionary and economically importantapplications for engineered tissues and organs exist in the human healtharena, the full economic potential of the industry is far from realized.At present, only two tissue engineering companies worldwide have beenable to commercialize cell based, skin substitute products focused oncutaneous wound healing and achieve annual sales in excess of $100million.

A major impediment to the acceptance of engineered tissues by medicalpractitioners, healthcare providers, and second party payers is the lackof a means to effectively and efficiently preserve and store engineeredtissues. The nature of living cells and tissue products makesdevelopment of long-term storage challenging. Current engineered tissuesmust often be stored and shipped under carefully controlled conditionsto maintain viability and function. Typically, engineered tissueproducts take weeks or months to produce but must be used within hoursor days after manufacture. As a result, TE companies must continuallyoperate with their production facilities at top capacity and absorb thecosts of unsold product which must be discarded. These inventory losses,on top of already costly manufacturing process, have forced prices toimpractical levels. As one specific example, APLIGRAF requires aboutfour weeks to manufacture, is usable for only 15 days and must bemaintained between 20 and 23° C. until used. As another example, EPICELis transported by a nurse from Genzyme Biosurgery's production facilityin Cambridge, Mass. to the point of use in a portable incubator and isused immediately upon arrival. Such constraints represent significantchallenges to developing convenient and cost-effective products.

Cryopreservation has been explored as a solution to the storage problem,but it is known to induce tissue damage through ice formation, chillinginjury, and osmotic imbalance. Besides APLIGRAF, the only other approvedliving skin equivalent, ORCEL, has been evaluated as a frozen productbut had the drawback that it must be maintained at temperatures below−100° C. prior to use. This requires specialized product delivery andstorage conditions, including the use of dangerous goods duringtransport, and use of liquid nitrogen for storage, which is expensive,dangerous, and not readily available in rural clinics and fieldhospitals.

Accordingly, what is needed in the art are improved methods ofcryopreserving viable engineered tissues and cells for storage underconditions that are routinely available at the point of use.

SUMMARY OF THE INVENTION

The present invention relates generally to systems and methods forcryopreservation of viable human skin substitutes.

In some embodiments, the present invention provides methods ofcryopreserving an organotypically cultured skin equivalent to maintainviable tissue comprising: treating an organotypically cultured skinequivalent in a cryoprotectant solution in a single step; packaging theorganotypically cultured skin equivalent to provide a packaged skinequivalent; and freezing the organotypically cultured skin equivalent toprovide a packaged cryopreserved skin equivalent. In some embodiments,the cryoprotectant is provided in a solution comprising about 20% or 21%to about 70% of the solution by volume, and more preferably about 20% or21% to about 45% of the solution by volume or 37.5% to 62.5% of thesolution by volume, or most preferably from about 25% to 40% of thesolution by volume or 42.5% to 57.5% of the solution by volume,depending on the temperature. In some embodiments, the treatment withcryoprotectant is conducted at from about 2 C to 8 C, while in otherembodiments, the treatment step is conducted at room temperature, forexample from about 15 C to 30 C. In some embodiments, the cryoprotectantis glycerol. In some embodiments, the freezing further comprisesfreezing the organotypically cultured skin equivalent in the absence ofsubstantial excess cryoprotectant. In some embodiments, the freezingfurther comprises freezing at about −80 C. In some embodiments, thefreezing further comprises direct exposure to temperatures ranging fromabout −50 C to −100 C. In some embodiments, the packaging furthercomprises enclosing the cryopreserved skin equivalent in a sterile bagand enclosing the sterile bag in a second bag. In some embodiments, theorganotypically cultured skin equivalents comprise NIKS cells. In someembodiments, the NIKS cells comprise an exogenous nucleic acid sequenceencoding an exogenous polypeptide. In some embodiments, the skinequivalent retains viability after thawing. In some embodiments, theskin equivalent has an A₅₅₀ of at least 50% of a reference skinequivalent as determined by an MTT assay.

In some embodiments, the methods further comprising thawing saidcryopreserved skin equivalent and applying said thawed skin equivalentto a patient in need thereof, wherein said thawed skin equivalent is notrinsed prior to said application to said patient. In some embodiments,the present invention provides methods of thawing a cryopreserved skinequivalent prior to application to a subject, comprising: warming thecryopreserved skin equivalent; and contacting the cryopreserved skinequivalent with a diffusion mediator comprising a tissue compatiblesolution to allow removal of the cryoprotectant solution by diffusion.In some embodiments, the warming comprises exposure to room temperatureat the site of use. In some embodiments, the diffusion mediator isselected from the group consisting of an absorbent medium, a membrane,and a dialysis bag. In some embodiments, the absorbent medium isselected from the group consisting of Telfa pads, foam pads, gauze pads,and cellulosic pads containing the tissue compatible medium. In someembodiments, the tissue compatible solution is a buffered solution.

In some embodiments, the present invention provides methods of treatinga subject comprising providing a packaged cryopreserved skin equivalentproduced as described above; aseptically transferring the cryopreservedskin equivalent from the package; warming the cryopreserved skinequivalent; contacting the cryopreserved skin equivalent with anabsorbent medium comprising a tissue compatible solution to allowremoval of the cryoprotectant solution by diffusion; and applying thecryopreserved skin equivalent to the subject.

In some embodiments, the present invention provides a cryopreserved skinequivalent equilibrated with a cryoprotectant, the skin equivalent beingsubstantially free of excess cryoprotectant on the exterior surface ofthe skin equivalent. In some embodiments, the present invention providesa system comprising the foregoing skin equivalent disposed on anabsorbent medium.

In some embodiments, the present invention provides methods comprisingproviding the packaged cryopreserved skin equivalent as described above;and applying the skin equivalent to a wound under conditions such thatthe skin equivalent contacts the wound.

In some embodiments, the present invention provides a kit comprising acryopreserved skin substitute, an absorbent medium, and a tissuecompatible solution. In some embodiments, the cryopreserved skinsubstitute is packaged in a sealable enclosure. In some embodiments, thecryopreserved skin substitute is provided in a culture vessel packagedin the bag.

In some embodiments, the present invention provides a method comprising:providing a culture dish comprising a cell culture substrate movablebetween defined upper and lower positions in the culture dish, forming askin equivalent on the cell culture substrate, wherein the cell culturesubstrate is at the upper position, lowering the cell culture substrateto the lower position for further processing. In some embodiments, thefurther processing comprises treating the skin equivalent with acryoprotectant solution. In some embodiments, the further processingcomprises freezing the skin equivalent in the culture dish.

In some embodiments, the present invention provides a method ofproducing a cryopreserved skin equivalent comprising: providing aculture dish comprising an insert movable between upper and lowerpositions in the culture dish, the insert having a bottom planar surfaceformed from a porous membrane, forming a dermal equivalent comprisingfibroblast cells on the porous membrane in the insert, wherein theinsert is placed the upper position the culture dish, culturing thefibroblast cells to form a dermal equivalent, applying keratinocytecells to the dermal equivalent, culturing the keratinocytes in a culturemedium under conditions such that the keratinocytes form a skinequivalent comprising stratified epithelium, removing the culturemedium, lowering the insert to the lower position, treating the skinequivalent with a cryoprotectant solution, and freezing the skinequivalent in the culture dish.

In some embodiments, the present invention provides methods of treatinga patient in need thereof with a cryopreserved skin equivalent made bythe foregoing methods comprising thawing said cryopreserved skinequivalent and applying said thawed skin equivalent to said patient inneed thereof, wherein said thawed skin equivalent is not rinsed prior tosaid application to said patient.

DESCRIPTION OF FIGURES

FIG. 1 is a graph of viability testing of thawed tissues using the MTTassay. The tissues were cryopreserved using a controlled-rate freezer(CRF) or by passive freezing in an ultra-cold freezer (set to −80° C.)and stored for up to 3 months, then thawed and analyzed at 1 hour and 1day. Viability was measured by MTT analysis of three 0.5 cm² punchesfrom each 44 cm² tissue at each time point (mean+/−1 st. dev.).

FIG. 2 is a temperature profile from a simulated passive tissue freeze.Temperature probes were affixed to the bottom of three 100 mm×20 mmdishes with a Transwell insert on top. The lid was placed on the dishand then they were packaged within an inner Whirl-pak pouch and an outerMylar pouch and placed in an ultra-cold freezer set to −80° C. Thepackaged tissues were placed in a metal rack at a bottom, middle, andtop position.

FIG. 3 is a graph of viability testing of thawed tissues using the MTTassay. The tissues were cryopreserved using a CRF and then stored inultra-cold freezers set to −80° C. or −50° C. for 1 to 6 months, thenthawed and analyzed at 1 hour and 1 day. Viability was measured by MTTanalysis of three 0.5 cm² punches from each 44 cm² tissue at each timepoint (mean+/−1 st. dev.).

FIG. 4 is a graph of viability testing of thawed tissues using the MTTassay. Tissues from two independent lots were cryopreserved and thenstored in an ultra-cold freezer set to −80° C. for greater than 1 month.Two tissues from each lot were placed into a container of dry ice for 48hours, with two control tissues from each lot remaining in theultra-cold freezer. At the end of the dry ice exposure, all tissues werethawed and analyzed at 15 minutes and 1 day. Viability was measured byMTT analysis of four 0.5 cm² punches from each 44 cm² tissue at eachtime point (mean+/−1 st. dev.).

FIG. 5 is a graph of viability testing of thawed tissues using the MTTassay. The tissues were cryopreserved using a CRF following a stepwisetemperature reduction (room temperature to 2-8° C. to −20° C.) duringwhich the tissues were exposed to a graded series of glycerolconcentrations soaked into sterile cellulose pads. All tissues wereinitially exposed to 16.3% glycerol at room temperature. Next, alltissues were switched to 2-8° C., with two tissues transferred to 32.5%glycerol. Lastly, all tissues were switched to −20° C., with one tissuetransferred to 65% glycerol. Tissues were stored in vapor-phase LN2 for6 days. Viability was measured by MTT analysis of three 0.5 cm² punchesfrom each 44 cm² tissue at 1 hour and 1 day post-thaw (mean+/−1 st.dev.).

FIG. 6 is a graph of viability testing of thawed tissues using the MTTassay. The tissues were cryopreserved in 65% glycerol by passivefreezing in an ultra-cold freezer set to −80° C. and then stored at −80°C. for 6 weeks. Tissues were thawed and placed in a hold chamber,consisting of two cellulose filter pads on a raised stainless steellifter, containing 100 ml of the specified solution and held for 1 hourat the specified temperature. Tissues were analyzed after 1 hour and 1day. Viability was measured by MTT analysis of three 0.5 cm² punchesfrom each 44 cm² tissue at each time point (mean+/−1 st. dev.).

FIG. 7 is a graph of viability testing of thawed tissues using the MTTassay. The tissues were cryopreserved in 65% glycerol and then stored invapor-phase LN₂ for 2 weeks. One tissue was thawed directly into agrowth chamber without a cellulose pad, while the other was placed in ahold chamber, consisting of 2 cellulose filter pads on a raisedstainless steel lifter, containing 100 ml of the growth media. Thethawed tissues were held for 1 hour at 37° C. Tissues were analyzedafter 1 hour and 1 day. Viability was measured by MTT analysis of three0.5 cm² punches from each 44 cm² tissue at each time point (mean+/−1 st.dev.).

FIG. 8 is a graph of viability testing of thawed tissues using the MTTassay. Tissues from one lot were cryopreserved and then stored in anultra-cold freezer set to −80° C. for 1 week. The tissues were thawedfor 10 minutes, placed in hold chambers containing either Telfa pads orWhatman pads soaked with 40 ml of a buffered nutrient solution warmed toeither 37° C. or room temperature (n=2 tissues per thaw condition). Thetissues were placed in the hold chambers for 15-20 minutes, and thenre-cultured in a growth chamber for 1 day. The tissues were analyzed at1 day post-thaw. Viability was measured by MTT analysis of four 0.5 cm²punches from each 44 cm² tissue (mean+/−1 st. dev.). The viabilityspecification is indicated by the red dashed line.

FIG. 9 is a graph of viability testing of thawed tissues using the MTTassay. Tissues from three independent lots were treated with 50%glycerol at 2 to 8° C. and cryopreserved as described in Example 2.Cryopreserved tissues were stored in an ultra-cold freezer atapproximately −80° C. for up to 12 months. After 0, 2, 3, 5, and 12months of storage, two tissues from each lot were thawed at roomtemperature and held on media saturated Telfa pads for 15 minutes.Tissues were then returned to culture overnight in a culture dish with araised stainless steel lifter, containing 90 ml of growth media.Viability was measured by MTT analysis of four 0.5 cm² punches from each44 cm² tissue at each time point (mean+/−1 st. dev.).

FIG. 10 is a graph of viability testing of thawed tissues using the MTTassay. Tissues from one lot were treated with the specified glycerolconcentration (32.5% or 50%) at the listed conditions prior to thefreeze and then stored in an ultra-cold freezer set to −80° C. for 9days. The tissues were thawed for 10 minutes, placed on a Telfa stacksoaked with 40 ml of a buffered nutrient solution for 15-20 minutes, andthen re-cultured in a growth chamber for 1 day. The tissues wereanalyzed at 1 day post-thaw. Viability was measured by MTT analysis offour 0.5 cm² punches from each 44 cm² tissue (mean+/−1 st. dev.). Theviability specification is indicated by the red dashed line.

DEFINITIONS

As used herein, the terms “skin equivalent”, “human skin equivalent”,“human skin substitute”, and “organotypic cultures” are usedinterchangeably to refer to an in vitro derived culture of keratinocytesthat has stratified into squamous epithelia. Typically, the skinequivalents are produced by organotypic culture and include a dermallayer in addition to a keratinocyte layer.

As used herein, the term “sterile” refers to a skin equivalent that isessentially or completely free of detectable microbial or fungalcontamination.

As used herein, the term “NIKS cells” refers to cells having thecharacteristics of the cells deposited as cell line ATCC CRL-1219. NIKSstands for near-diploid immortalized keratinocytes.

As used herein, the term “viable” when used in reference to a skinequivalent refers to the viability of cells in the skin equivalentfollowing cryopreservation. In preferred embodiments, a “viable” skinhas an A₅₅₀ of at least 50%, 60%, 70%, 80% or 90% of a controlnon-cryopreserved tissue as measured by an MTT assay or at least 50%,60%, 70%, 80% or 90% of the readout value of a similar viability assay.

DETAILED DESCRIPTION

The present invention relates generally to systems and methods forcryopreservation of human skin substitutes. In particular, the presentinvention relates to methods for cryopreserving viable human skinequivalents so that they can be stored for prolonged periods at the siteof use, such as a hospital, operating room or burn unit. The methodsdisclosed herein allow for novel increases in efficiency of thepreservation process and utilization of preserved skin equivalents,including single-step equilibration in a cryoprotectant, packaging ofthe skin equivalent in a sterile package prior to cryropreservation, andthe ability to use the cryopreserved tissues for direct application to apatient (e.g., in a grafting procedure) without rinsing. In someembodiments, the present invention provides ready-to-use cryopreservedskin equivalents for use in treatment of a patient, and in preferredembodiments for use in grafting procedures. The cryopreserved skinequivalent is designed for long term storage at the site of use. In someembodiments, the cryopreserved equivalents are engineered to deliver thebroad spectrum human host defense peptides such as β-defensin-3 (hBD-3)or cathelicidin (hCAP18/LL-37), or pro-angiogenic factors, to the woundbed.

Previously, cadaver skin has been harvested and cryopreserved bytreatment with from 10% to 20% glycerol as a cryopreservative. See e.g.,Kagan et al., Clin Lab Med 25 (2005) 587-605. Surprisingly, it has beenfound that increased glycerol concentrations are needed to cryopreservehuman skin equivalents.

Accordingly, in some embodiments, the present invention provides acryopreserved skin equivalent. In some embodiments, the skin equivalenthas been engineered to express and provide exogenous antimicrobialpolypeptides or pro-angiogenic factors. The present invention is notlimited to the use of any particular antimicrobial polypeptide. Inpreferred embodiments, the antimicrobial polypeptide is humanβ-defensin-1, human β-defensin-2, human β-defensin-3, or cathelicidin(hCAP-18/LL37) or variant. In some preferred embodiments, nucleic acidconstructs or vectors encoding the antimicrobial polypeptide orpro-angiogenic factor are introduced into the keratinocytes (e.g., NIKScells) and the transfected keratinocytes are used to make the skinequivalent by organotypic culture techniques. Preferred embodiments forthe production of skin equivalents expressing exogenous polypeptides, aswell as additional wild-type and variant antimicrobial polypeptides canbe found in U.S. Pat. Nos. 7,674,291; 7,807,148; 7,915,042; 7,988,959;and 8,092,531; each of which is incorporated herein by reference in itsentirety.

In some embodiments, the cryopreserved skin equivalents are applied towounds after thawing and left in place. In other embodiments, thecryopreserved skin equivalents are applied temporarily to wounds. Insome embodiments, the cryopreserved skin equivalents are removed andreplaced with additional cryopreserved human skin equivalents.

A) Skin Equivalents Produced by Organotypic Culture

The present invention is not limited to the use of any particular sourceof cells that are capable of differentiating into squamous epithelia.Indeed, the present invention contemplates the use of a variety of celllines and sources that can differentiate into squamous epithelia,including both primary and immortalized keratinocytes. Sources of cellsinclude keratinocytes and dermal fibroblasts biopsied from humans andcavaderic donors (Auger et al., In Vitro Cell. Dev. Biol.—Animal36:96-103; U.S. Pat. Nos. 5,968,546 and 5,693,332, each of which isincorporated herein by reference), neonatal foreskins (Asbill et al.,Pharm. Research 17(9): 1092-97 (2000); Meana et al., Burns 24:621-30(1998); U.S. Pat. Nos. 4,485,096; 6,039,760; and 5,536,656, each ofwhich is incorporated herein by reference), and immortalizedkeratinocytes cell lines such as NM1 cells (Baden, In Vitro Cell. Dev.Biol. 23(3):205-213 (1987)), HaCaT cells (Boucamp et al., J. cell. Boil.106:761-771 (1988)); and NIKS cells (Cell line BC-1-Ep/SL; U.S. Pat. No.5,989,837, incorporated herein by reference; ATCC CRL-12191). Each ofthe mentioned cell lines can be cultured or genetically modified inorder to produce a cell line capable of expressing or co-expressing thedesired protein(s). In particularly preferred embodiments, NIKS cellsare utilized. The discovery of the novel NIKS human keratinocyte cellline provides an opportunity to genetically engineer human keratinocyteswith non-viral vectors. A unique advantage of the NIKS cells is thatthey are a consistent source of genetically-uniform, pathogen-free humankeratinocytes. For this reason, they are useful for the application ofgenetic engineering and genomic gene expression approaches to providehuman skin equivalents with enhanced properties over currently availableskin equivalents. NIKS cells, identified and characterized at theUniversity of Wisconsin, are nontumorigenic, karyotypically stable, andexhibit normal growth and differentiation both in monolayer andorganotypic culture. NIKS cells form fully stratified skin equivalentsin culture. These cultures are indistinguishable by all criteria testedthus far from organotypic cultures formed from primary humankeratinocytes. Unlike primary cells however, NIKS cells exhibit anextended lifespan in monolayer culture. This provides an opportunity togenetically manipulate the cells and isolate new clones of cells withnew useful properties (Allen-Hoffmann et al., J. Invest. Dermatol.,114(3): 444-455 (2000)).

The NIKS cells arose from the BC-1-Ep strain of human neonatal foreskinkeratinocytes isolated from an apparently normal male infant. In earlypassages, the BC-1-Ep cells exhibited no morphological or growthcharacteristics that were atypical for cultured normal humankeratinocytes. Cultivated BC-1-Ep cells exhibited stratification as wellas features of programmed cell death. To determine replicative lifespan,the BC-1-Ep cells were serially cultivated to senescence in standardkeratinocyte growth medium at a density of 3×10⁵ cells per 100-mm dishand passaged at weekly intervals (approximately a 1:25 split). Bypassage 15, most keratinocytes in the population appeared senescent asjudged by the presence of numerous abortive colonies which exhibitedlarge, flat cells. However, at passage 16, keratinocytes exhibiting asmall cell size were evident. By passage 17, only the small-sizedkeratinocytes were present in the culture and no large, senescentkeratinocytes were evident. The resulting population of smallkeratinocytes that survived this putative crisis period appearedmorphologically uniform and produced colonies of keratinocytesexhibiting typical keratinocyte characteristics including cell-celladhesion and apparent squame production. The keratinocytes that survivedsenescence were serially cultivated at a density of 3×10⁵ cells per100-mm dish. Typically the cultures reached a cell density ofapproximately 8×10⁶ cells within 7 days. This stable rate of cell growthwas maintained through at least 59 passages, demonstrating that thecells had achieved immortality. The keratinocytes that emerged from theoriginal senescencing population are now termed NIKS. The NIKS cell linehas been screened for the presence of proviral DNA sequences for HIV-1,HIV-2, EBV, CMV, HTLV-1, HTLV-2, HBV, HCV, B-19 parvovirus, HPV-16,SV40, HHV-6, HHV-7, HPV-18 and HPV-31 using either PCR or Southernanalysis. None of these viruses were detected.

Chromosomal analysis was performed on the parental BC-1-Ep cells atpassage 3 and NIKS cells at passages 31 and 54. The parental BC-1-Epcells have a normal chromosomal complement of 46, XY. At passage 31, allNIKS cells contained 47 chromosomes with an extra isochromosome of thelong arm of chromosome 8. No other gross chromosomal abnormalities ormarker chromosomes were detected. The karyotype of the NIKS cells hasbeen shown to be stable to at least passage 54.

The DNA fingerprints for the NIKS cell line and the BC-1-Epkeratinocytes are identical at all twelve loci analyzed demonstratingthat the NIKS cells arose from the parental BC-1-Ep population. The oddsof the NIKS cell line having the parental BC-1-Ep DNA fingerprint byrandom chance is 4×10⁻¹⁶. The DNA fingerprints from three differentsources of human keratinocytes, ED-1-Ep, SCC4 and SCC13y are differentfrom the BC-1-Ep pattern. This data also shows that keratinocytesisolated from other humans, ED-1-Ep, SCC4, and SCC13y, are unrelated tothe BC-1-Ep cells or each other. The NIKS DNA fingerprint data providesan unequivocal way to identify the NIKS cell line.

Loss of p53 function is associated with an enhanced proliferativepotential and increased frequency of immortality in cultured cells. Thesequence of p53 in the NIKS cells is identical to published p53sequences (GenBank accession number: M14695). In humans, p53 exists intwo predominant polymorphic forms distinguished by the amino acid atcodon 72. Both alleles of p53 in the NIKS cells are wild-type and havethe sequence CGC at codon 72, which codes for an arginine. The othercommon form of p53 has a proline at this position. The entire sequenceof p53 in the NIKS cells is identical to the BC-1-Ep progenitor cells.Rb was also found to be wild-type in NIKS cells.

Anchorage-independent growth is highly correlated to tumorigenicity invivo. For this reason, the anchorage-independent growth characteristicsof NIKS cells in agar or methylcellulose-containing medium wereinvestigated. NIKS cells remained as single cells after 4 weeks ineither agar- or methylcellulose-containing medium. The assays werecontinued for a total of 8 weeks to detect slow growing variants of theNIKS cells. None were observed.

To determine the tumorigenicity of the parental BC-1-Ep keratinocytesand the immortal NIKS keratinocyte cell line, cells were injected intothe flanks of athymic nude mice. The human squamous cell carcinoma cellline, SCC4, was used as a positive control for tumor production in theseanimals. The injection of samples was designed such that animalsreceived SCC4 cells in one flank and either the parental BC-1-Epkeratinocytes or the NIKS cells in the opposite flank. This injectionstrategy eliminated animal to animal variation in tumor production andconfirmed that the mice would support vigorous growth of tumorigeniccells. Neither the parental BC-1-Ep keratinocytes (passage 6) nor theNIKS keratinocytes (passage 35) produced tumors in athymic nude mice.

NIKS cells were analyzed for the ability to undergo differentiation inboth submerged culture and organotypic culture. Techniques fororganotypic culture are described in detail in the examples. Inparticularly preferred embodiments, the organotypically cultured skinequivalents of the present invention comprise a dermal equivalent formedfrom collagen or a similar material and fibroblasts. The keratinocytes,for example NIKS cells or a combination of NIKS cells and cells from apatient are seeded onto the dermal equivalent and form an epidermallayer characterized by squamous differentiation following theorganotypic culture process.

For cells in submerged culture, the formation cornified envelopes wasmonitored as a marker of squamous differentiation. In cultured humankeratinocytes, early stages of cornified envelope assembly result in theformation of an immature structure composed of involucrin, cystatin-αand other proteins, which represent the innermost third of the maturecornified envelope. Less than 2% of the keratinocytes from the adherentBC-1-Ep cells or the NIKS cell line produce cornified envelopes. Thisfinding is consistent with previous studies demonstrating that activelygrowing, subconfluent keratinocytes produce less than 5% cornifiedenvelopes. To determine whether the NIKS cell line is capable ofproducing cornified envelopes when induced to differentiate, the cellswere removed from adherent culture and suspended for 24 hours in mediummade semi-solid with methylcellulose. Many aspects of terminaldifferentiation, including differential expression of keratins andcornified envelope formation can be triggered in vitro by loss ofkeratinocyte cell-cell and cell-substratum adhesion. The NIKSkeratinocytes produced as many as and usually more cornified envelopesthan the parental keratinocytes. These findings demonstrate that theNIKS keratinocytes are not defective in their ability to initiate theformation of this cell type-specific differentiation structure.

To confirm that the NIKS keratinocytes can undergo squamousdifferentiation, the cells were cultivated in organotypic culture.Keratinocyte cultures grown on plastic substrata and submerged in mediumreplicate but exhibit limited differentiation. Specifically, humankeratinocytes become confluent and undergo limited stratificationproducing a sheet consisting of 3 or more layers of keratinocytes. Bylight and electron microscopy there are striking differences between thearchitecture of the multilayered sheets formed in submerged culture andintact human skin. In contrast, organotypic culturing techniques allowfor keratinocyte growth and differentiation under in vivo-likeconditions. Specifically, the cells adhere to a physiological substratumconsisting of dermal fibroblasts embedded within a fibrillar collagenbase. The organotypic culture is maintained at the air-medium interface.In this way, cells in the upper sheets are air-exposed while theproliferating basal cells remain closest to the gradient of nutrientsprovided by diffusion through the collagen gel. Under these conditions,correct tissue architecture is formed. Several characteristics of anormal differentiating epidermis are evident. In both the parental cellsand the NIKS cell line a single layer of cuboidal basal cells rests atthe junction of the epidermis and the dermal equivalent. The roundedmorphology and high nuclear to cytoplasmic ratio is indicative of anactively dividing population of keratinocytes. In normal humanepidermis, as the basal cells divide they give rise to daughter cellsthat migrate upwards into the differentiating layers of the tissue. Thedaughter cells increase in size and become flattened and squamous.Eventually these cells enucleate and form cornified, keratinizedstructures. This normal differentiation process is evident in the upperlayers of both the parental cells and the NIKS cells. The appearance offlattened squamous cells is evident in the upper epidermal layers anddemonstrates that stratification has occurred in the organotypiccultures. In the uppermost part of the organotypic cultures theenucleated squames peel off the top of the culture. To date, nohistological differences in differentiation at the light microscopelevel between the parental keratinocytes and the NIKS keratinocyte cellline grown in organotypic culture have been observed.

To observe more detailed characteristics of the parental (passage 5) andNIKS (passage 38) organotypic cultures and to confirm the histologicalobservations, samples were analyzed using electron microscopy. Parentalcells and the immortalized NIKS human keratinocyte cell line wereharvested after 15 days in organotypic culture and sectionedperpendicular to the basal layer to show the extent of stratification.Both the parental cells and the NIKS cell line undergo extensivestratification in organotypic culture and form structures that arecharacteristic of normal human epidermis. Abundant desmosomes are formedin organotypic cultures of parental cells and the NIKS cell line. Theformation of a basal lamina and associated hemidesmosomes in the basalkeratinocyte layers of both the parental cells and the cell line wasalso noted.

Hemidesmosomes are specialized structures that increase adhesion of thekeratinocytes to the basal lamina and help maintain the integrity andstrength of the tissue. The presence of these structures was especiallyevident in areas where the parental cells or the NIKS cells had attacheddirectly to the porous support. These findings are consistent withearlier ultrastructural findings using human foreskin keratinocytescultured on a fibroblast-containing porous support. Analysis at both thelight and electron microscopic levels demonstrate that the NIKS cellline in organotypic culture can stratify, differentiate, and formstructures such as desmosomes, basal lamina, and hemidesmosomes found innormal human epidermis.

B) Cryopreservation

The present invention provides cryopreserved viable skin equivalents.The cryopreserved skin equivalents are preferable storable atapproximately −50 C, −60 C, −70 C, −80 C or colder for an extendedperiod of time such as greater than 1, 2, 3, 4, 5 or 6 months and up to12 or 24 months without a substantial loss of viability.

In preferred embodiments, all steps of the cryopreservation processprior to product packaging are performed aseptically inside a Class 100biosafety cabinet in a Class 10,000 cleanroom. In some embodiments, thecryopreservation process comprises treating an organotypically culturedskin equivalent in a cryoprotectant solution. Certain embodiments of thepresent invention are not limited to the use of any particularcryoprotectant. In some preferred embodiments, the cryoprotectant isglycerol. The cryoprotectant may be provided in different concentrationsin the cryoprotectant solution. In some embodiments, the cryoprotectantis provided in a solution comprising about 20% or 21% to about 70% ofthe solution by volume, and more preferably about 20% or 21% to about45% of the solution by volume or 37.5% to 62.5% of the solution byvolume, or most preferably from about 25% to 40% of the solution byvolume or 42.5% to 57.5% of the solution by volume, depending on thetemperature. In some embodiments, the cryoprotectant solution preferablycomprises about 32.5% v/v or about 50% v/v cryoprotectant (e.g.,glycerol). In some embodiments, the cryoprotectant is provided in a basemedium solution. Suitable base medium solutions include, but are notlimited to, DMEM, Ham's F-10, Ham's F-12, DMEM/F-12, Medium 199, MEM andRPMI. In some embodiments, the base medium forms the remainder of thesolution volume. In some embodiments, the cryoprotectant solution isbuffered. Suitable buffers include, but are not limited to, HEPES, Tris,MOPS, and Trizma buffers. Buffering agents may be included at an amountto provide a buffered system in the range of pH 7.0 to 7.4. In somepreferred embodiments, the cryoprotectant solution is buffered with fromabout 5 mM to 15 mM HEPES, most preferably about 10 mM HEPES to a pH ofabout 7.0 to 7.4.

In some particularly preferred embodiments, treatment with thecryoprotectant solution is conducted in a single step. By “single step”it is meant that the cryoprotectant solution is not exchanged during theequilibration procedure as is common in the art. For example, thetreatment step is performed using a cryoprotectant solution with adefined concentration of cryoprotectant as opposed to a stepwiseequilibration procedure where several media changes with increasingconcentrations of cryoprotectant at each step. In some embodiments, thetreatment step is conducted at a reduced temperature. In preferredembodiments, the treatment step is conducted at from about 2 C to 8 C,while in other embodiments, the treatment step is conducted at roomtemperature, for example from about 15 C to 30 C. In some embodiments,the skin equivalent is incubated in the cryoprotectant solution forabout 10 to 60 minutes, preferably from about 20 to 30 minutes.

In some embodiments, the skin equivalent is frozen following treatmentwith the cryoprotectant solution. In some embodiments, excesscryoprotectant solution is removed from the skin equivalent prior tofreezing, for example by aspirating the solution or moving the treatedskin equivalent to a fresh vessel. Accordingly, in some embodiments, thetreated skin equivalent is frozen by exposure to temperatures rangingfrom about −50 C to −100 C, and most preferably at about −80 C. In somepreferred embodiments treated the skin equivalent is simply placed in abag or other vessel such as a culture dish and placed in a freezing unitsuch as a low temperature (e.g., −80 C freezer) freezing unit. Incontrast, it is common in the art to control the rate of freezing eitherby controlling the temperature in the freezing unit or by placing thetissue to be frozen in a container that allows control of the rate ofdecrease in temperature. In some embodiments, the treated skinequivalent is placed in a sterile culture vessel for freezing. The term“culture vessel” refers to any vessel of the type commonly used toculture cells or tissues and include circular, rectangular, and squaredishes formed from a suitable material such as tissue culture plastic,polystyrene, polymers, plastics, glass, etc.

In some embodiments, the cryopreserved skin equivalent is packaged forlong term storage. In some preferred embodiments, the skin equivalent isplaced in a bag or culture vessel as described above. In someembodiments, the bag or culture vessel is sealed, preferably heat sealedin a sterile bag (e.g., a plastic or polymer bag) to provide a primarypackage. The primary package is then sealed inside a secondary bag, forexample a secondary plastic, foil, or Mylar bag. The cryopreservedtissues of the present invention may preferably be stored at lowtemperature, from about −50 C to about −100 C, preferably about −80 C.The skin equivalents may be preferably stored from about 1, 2, 3, 4, 5or 6 months and up to 12 or 24 months without a substantial loss ofviability.

In some embodiments, the present invention provides a method of thawinga cryopreserved skin equivalent prior to application to a subject,comprising warming said cryopreserved skin equivalent and contactingsaid cryopreserved skin equivalent with an absorbent medium comprising atissue compatible solution to allow removal of said cryoprotectantsolution by diffusion. In some embodiments, the cryopreserved skinequivalent in a suitable bag or culture vessel is simply placed on abench or table top and allowed to thaw. Thawing under controlledconditions as is common in the art is not necessary. In someembodiments, cryopreserved skin equivalent is placed on an absorbentmedium to remove thawed cryoprotectant solution from the skinequivalent. The present invention is not limited to the use a particularabsorbent medium. Suitable absorbent media include, but are not limitedto, Telfa pads, cellulosic pads (e.g., Whatman 1003-090 filter pads andPall 70010 filter pads), gauze pads, and foam pads (e.g., Covidien 55544hydrophilic foam pad). In some preferred embodiments, the absorbentmedium is a Telfa pad. In some embodiments, the absorbent mediumcomprises a tissue-compatible solution. In some embodiments, the tissuecompatible solution is a buffered solution. Suitable tissue compatiblesolutions include, but are not limited to, DMEM, Ham's F-10, Ham's F-12,DMEM/F-12, Medium 199, MEM and RPMI. Suitable buffers include, but arenot limited to, HEPES, Tris, MOPS, and Trizma buffers. Buffering agentsmay be included at an amount to provide a buffered system in the rangeof pH 7.0 to 7.4.

In further embodiments, the present invention provides kits comprising acryopreserved skin substitute, preferably provided in a package asdescribed above. In some embodiments, the kits further comprise anabsorbent medium, and a tissue compatible solution.

In some embodiments, the present invention provides a process forforming an organotypically cultured skin equivalent and freezing theskin equivalent in the same culture vessel. In some embodiments, theculture vessel comprises an insert movable between upper and lowerpositions in the culture dish. The insert preferably comprises a bottomsurface which is a porous membrane. In use, the vessel is filled withthe appropriate culture medium and a dermal equivalent is formed on theporous membrane. The dermal equivalent is then seeded with keratinocytes(e.g., NIKS cells) in the presence of the appropriate culture medium. Atthe appropriate time, an air interface is created by lowering the levelof culture medium in the vessel and the culture is continued until thestratified skin equivalent is formed. The culture medium is then removedfrom the vessel and the insert is lowered to the lower position. Thecryoprotectant solution is added for treatment and then removed, and thevessel is then transferred to the freezing unit.

C) Therapeutic Uses

It is contemplated that the cryopreserved skin equivalents of thepresent invention may be used therapeutically. In some embodiments, thecryopreserved skin substitute is used in wound closure and burntreatment applications. The use of autografts and allografts for thetreatment of burns and wound closure is described in Myers et al., A. J.Surg. 170(1):75-83 (1995) and U.S. Pat. Nos. 5,693,332; 5,658,331; and6,039,760, each of which is incorporated herein by reference. In someembodiments, the skin equivalents may be used in conjunction with dermalreplacements such as DERMAGRAFT or INTEGRA. Accordingly, the presentinvention provides methods for wound closure, including ulcers or woundscaused by burns, comprising providing a skin equivalent and a patientsuffering from a wound and treating the patient with the skin equivalentunder conditions such that the wound is closed.

In some embodiments, the skin equivalents are utilized to treat chronicskin wounds. Chronic skin wounds (e.g., venous ulcers, diabetic ulcers,pressure ulcers) are a serious problem. The healing of such a woundoften takes well over a year of treatment. Treatment options currentlyinclude dressings and debridement (use of chemicals or surgery to clearaway necrotic tissue), and/or antibiotics in the case of infection.These treatment options take extended periods of time and high levels ofpatient compliance. As such, a therapy that can increase apractitioner's success in healing chronic wounds and accelerate the rateof wound healing would meet an unmet need in the field. Accordingly, thepresent invention contemplates treatment of skin wounds withcryopreserved skin equivalents. In some embodiments, skin equivalentsare topically applied to wounds. In other embodiments, cryopreservedskin equivalents are used for application to partial thickness wounds.In other embodiments, cryopreserved skin equivalents are used to treatfull thickness wounds. In other embodiments, cryopreserved skinequivalents are used to treat numerous types of internal wounds,including, but not limited to, internal wounds of the mucous membranesthat line the gastrointestinal tract, ulcerative colitis, andinflammation of mucous membranes that may be caused by cancer therapies.In still other embodiments, skin equivalents expressing host defensepeptides or pro-angiogenic factors are used as a temporary or permanentwound dressing.

In still further embodiments, the cells are engineered to provideadditional therapeutic agents to a subject. The present invention is notlimited to the delivery of any particular therapeutic agent. Indeed, itis contemplated that a variety of therapeutic agents may be delivered tothe subject, including, but not limited to, enzymes, peptides, peptidehormones, other proteins, ribosomal RNA, ribozymes, small interferingRNA (siRNA) micro RNA (miRNA), and antisense RNA. In preferredembodiments, the agents are host defense peptides such as humanbeta-defensin 1, 2, or 3 or cathelicidin or other proteins such as VEGFand HIF-1α, see, e.g., U.S. Pat. Nos. 7,674,291; 7,807,148; 7,915,042;7,988,959; and 8,092,531; each of which is incorporated herein byreference in its entirety. These therapeutic agents may be delivered fora variety of purposes, including but not limited to the purpose ofcorrecting genetic defects. In some particular preferred embodiments,the therapeutic agent is delivered for the purpose of detoxifying apatient with an inherited inborn error of metabolism (e.g.,aminoacidopathesis) in which the skin equivalent serves as wild-typetissue. It is contemplated that delivery of the therapeutic agentcorrects the defect. In some embodiments, the cells are transfected witha DNA construct encoding a therapeutic agent (e.g., insulin, clottingfactor IX, erythropoietin, etc.) and skin equivalents prepared fromtransfected cells are administered to the subject. The therapeutic agentis then delivered to the patient's bloodstream or other tissues from thegraft. In preferred embodiments, the nucleic acid encoding thetherapeutic agent is operably linked to a suitable promoter. The presentinvention is not limited to the use of any particular promoter. Indeed,the use of a variety of promoters is contemplated, including, but notlimited to, inducible, constitutive, tissue-specific, andkeratinocyte-specific promoters. In some embodiments, the nucleic acidencoding the therapeutic agent is introduced directly into thekeratinocytes (i.e., by electroporation, calcium phosphateco-precipitation, or liposome transfection). In other preferredembodiments, the nucleic acid encoding the therapeutic agent is providedas a vector and the vector is introduced into the keratinocytes bymethods known in the art. In some embodiments, the vector is an episomalvector such as a replicating plasmid. In other embodiments, the vectorintegrates into the genome of the keratinocytes. Examples of integratingvectors include, but are not limited to, retroviral vectors,adeno-associated virus vectors, non-replicating plasmid vectors andtransposon vectors.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); mM (millimolar); μM(micromolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml or mL (milliliters);μL or μL (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); C (degrees Centigrade); U (units), mU(milliunits); min. (minutes); sec. (seconds); % (percent); kb(kilobase); by (base pair); PCR (polymerase chain reaction); BSA (bovineserum albumin); CFU (colony forming units); kGy (kiloGray); PVDF(polyvinylidine fluoride); BCA (bicinchoninic acid); SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis).

Example 1

StrataGraft® skin tissue is a living, full-thickness, allogeneic humanskin substitute that reproduces many of the structural and biologicalproperties of normal human skin. StrataGraft® skin tissue contains botha viable, fully-stratified epidermal layer derived from NIKS® cells,which are a consistent and well-characterized source of pathogen-freehuman keratinocyte progenitors, and a dermal layer containing normalhuman dermal fibroblasts (NHDF) embedded in a collagen-rich matrix.StrataGraft® skin tissue possesses excellent tensile strength andhandling characteristics that enable it to be meshed, stapled, andsutured similarly to human skin grafts. StrataGraft® also exhibitsbarrier function comparable to that of intact human skin and is capableof delivering bioactive molecules for wound bed conditioning and tissueregeneration. The physical and biological characteristics ofStrataGraft® skin tissue make it ideal for the treatment of a variety ofskin wounds.

The manufacturing process for StrataGraft® skin tissue encompasses threesequential cell and tissue culture processes. In Stage I of themanufacturing process, NIKS keratinocytes are expanded in monolayer cellculture. Concurrent with the NIKS keratinocyte culture in Stage I, NHDFare expanded in monolayer culture and combined with purified type Icollagen and culture medium and allowed to gel to form the cellularizeddermal equivalent (DE). Alternatively, NHDF are seeded into Transwellinserts and allowed to proliferate and secrete and assembleextracellular matrix molecules into a simplified dermal equivalent. InStage II, NIKS keratinocytes are seeded onto the surface of the DE andcultured under submerged conditions for two days to promote completeepithelialization of the DE surface. The tissue is then lifted to theair-liquid interface in Stage III, where it is maintained for 18 days ina controlled, low humidity environment to promote tissue maturation. Theskin equivalents are generally prepared as described in U.S. Pat. Nos.7,674,291; 7,807,148; 7,915,042; 7,988,959; and 8,092,531; each of whichis incorporated herein by reference in its entirety.

Example 2

This example describes improved cryopreservation methods for human skinequivalents. The production process is unchanged from the current methoddescribed above. All tissues in the lot are fed with fresh medium andincubated overnight prior to cryopreservation. Prior tocryopreservation, media samples from all tissues in each lot are testedfor sterility. The remaining tissues in each lot are cryopreserved asfollows.

Parameter Operating Range Cryoprotectant formulation 50% (v/v) glycerolDMEM (1X) 10 mM HEPES (pH 7.0 to 7.4) Pre-freeze cryoprotectantincubation 2-8° C. temperature Pre-freeze cryoprotectant 20-30 minutesincubation time Freeze method Direct transfer to −80° C. freezer Storagetemperature −70 to −90° C. Shipping conditions Overnight delivery on dryiceCryopreservation Process Description

All steps of the cryopreservation process prior to the final productpackaging step are performed aseptically inside a Class 100 biosafetycabinet in a Class 10,000 cleanroom.

-   Step 1—Pre-cool 100 mm culture dishes containing 20 ml of    cryoprotectant solution to 2-8° C. on a stainless steel cold    treatment surface inside biosafety cabinet. Temperature of cold    treatment surface is maintained at 2-8° C. for several hours by    contact with frozen gel packs submerged in water.-   Step 2—Transfer Transwells containing StrataGraft® tissues into    individual dishes containing pre-cooled cryoprotectant solution.    Incubate tissues 20-30 minutes in cryoprotectant on the cold    treatment surface.-   Step 3—Transfer treated StrataGraft® tissues to new sterile 100 mm    culture dishes containing final product label so that the tissue    rests on the bottom of the culture dish and return tissues back to    the cold treatment surface. Excess cryoprotectant is allowed to    drain from the skin equivalent to provide a treated skin equivalent    that is substantially free of excess cryoprotectant on the exterior    surfaces of the skin equivalent.-   Step 4—Heat-seal 100 mm culture dishes in clear, sterile bags. Place    primary package into secondary Mylar bag, heat-seal, and transfer    packaged tissues to cold storage container until all tissues are    packaged.-   Step 5—Remove cold storage container with packaged StrataGraft®    tissues from cleanroom and transfer tissues to an ultralow freezer    (−75° C. to −80° C.). Place tissues in a pre-cooled rack in the    freezer that allows unrestricted airflow to the top and bottom of    the packaged tissues to ensure uniform and rapid cooling. Leave    tissues undisturbed overnight during the freezing process.

Tissues are placed into quarantine storage at −70 to −90° C. pendingresults of lot release testing. A representative tissue from each lot ofcryopreserved StrataGraft® skin tissue is tested using a panel ofQuality Control SOP that have historically been used for lot releasetesting of StrataGraft® tissue.

Stratatech has established and qualified a panel of lot release assaysthat are used to characterize StrataGraft® skin tissue. A subset ofthese lot release assays has been used to monitor and evaluate theimpact that changes to the storage conditions may have on key biologicaland structural characteristics of StrataGraft® skin tissue (e.g.,barrier function, viability, and histological appearance). Althoughtransient minor changes in the histological appearance of StrataGraft®tissue are generally observed following cryopreservation, thehistological architecture normalizes after being reintroduced intoorganotypic culture for several days, indicating that the viable cellsin the basal layer of StrataGraft® are able to proliferate and reproducethe epidermal layer after cryopreservation. The systematic evaluation ofcryoprotectant concentration, incubation time, incubation temperature,freezing rate, and storage conditions has enabled Stratatech to identifya cryopreservation process that enables long-term storage ofStrataGraft® skin tissue with consistent and defined quality and thatmeets the specifications that define StrataGraft® skin tissue.

The following parameters were systematically evaluated duringdevelopment of the StrataGraft cryopreservation process.

-   -   Cryoprotectant composition    -   Pre-freeze cryoprotectant incubation temperature    -   Pre-freeze cryoprotectant incubation time    -   Number of steps required during cryoprotectant incubation    -   Freezing rate    -   Final product packaging    -   Storage temperature    -   Shipping conditions    -   Thaw temperature and time    -   Post-thaw cryoprotectant diffusion mediator    -   Post-thaw incubation solution    -   Post-thaw incubation temperature    -   Post-thaw incubation time

As anticipated, many of these individual parameters interact toinfluence the properties of cryopreserved tissue. For example, it is notpossible to optimize cryoprotectant concentration without also takinginto account the cryoprotectant incubation time and temperature.Likewise, post-thaw incubation temperature influences the allowablerange of post-thaw incubation times. During development of thecryopreservation process, a range of acceptable values for each of theindividual parameters was identified and used to define the finalcombination of cryoprotectant formulation, pre-freeze incubation time,pre-freeze incubation temperature, freeze rate, and storage condition.The operating parameters of the cryopreservation process as developedfor cryopreservation and storage of StrataGraft® skin tissue are listedabove.

Glycerol (glycerin) was identified as the most desirable cryoprotectantfor StrataGraft® tissue. The glycerol used in the cryoprotectantformulation is synthetic, USP-grade material that undergoes additionaltesting for endotoxin prior to release for use. In addition to glycerol,the cryoprotectant solution contains Dulbecco's Modified Eagle Medium(DMEM) and 10 mM HEPES to maintain pH of 7.0 to 7.4 at ambientatmospheric conditions. DMEM was chosen as the base for thecryopreservation solution because it is already a component of theculture medium used to prepare StrataGraft® skin tissue. HEPES is awell-characterized buffering agent that maintains the pH of thecryopreservation solution outside of a CO₂ environment.

A series of studies were performed to determine an appropriate glycerolconcentration for use in the cryoprotectant solution. Glycerolconcentrations tested ranged from 16.25% to 65%. In some cases, theconcentration of glycerol was gradually increased in two or three stepsby incubation in a series of solutions with increasing glycerolconcentration. Initial studies to demonstrate the feasibility ofcryopreserving StrataGraft® tissue used a three-step process in whichthe glycerol concentration was sequentially increased to 16.25%, then32.5%, and finally to 65% by incubation for 15-20 minutes in eachglycerol solution, while gradually reducing the temperature at eachincubation step (16.25% glycerol incubation at room temperature, 32.5%at 2-8° C., and 65% at −20° C.). Finally, tissues were frozen to −140°C. at −15° C./min in a controlled-rate freezer. Cryopreserved tissueswere transferred to an ultra-cold freezer (−80° C.), where they weremaintained until thawed for analysis. While this three-step processcould be used to preserve the viability and histological architecture ofStrataGraft® tissues during cryopreserved storage, the complexity ofhaving multiple steps with different solutions performed at differenttemperatures introduces the opportunity for error and is not amenable toprocess scale-up that would be required for commercialization.

Analysis of tissues cryopreserved with this three-step method revealedthat, after thawing and dilution of the cryoprotectant, the tissuesexhibited viability (assessed by the ability of viable cells in thetissue to convert MTT to its formazan product), histologicalarchitecture, and barrier function comparable to tissues that were notcryopreserved. The tissues maintained high levels of viability followingre-introduction into organotypic culture for up to nine days afterthawing, demonstrating that the metabolic activity detected shortlyafter thawing was not just residual enzymatic activity.

During these initial studies, it was found that the concentration ofglycerol in cryopreserved tissues could be reduced after thawing eitherby incubating the tissues in a series of solutions with decreasingglycerol concentration, or by placing the tissues in a media reservoirwith a filter pad just below the tissue to moderate the diffusion ofglycerol. Based on these results, subsequent studies primarily used thesingle-step approach of incubating thawed tissues in culture medium witha pad to moderate glycerol diffusion. See FIG. 7.

After demonstrating the feasibility of cryopreserving StrataGraft® skintissue using the three-step method, we used this method as a benchmarkagainst which to compare simplifications to the cryopreservation method.These studies examined reducing the number of steps required to reachthe same final glycerol concentration (65%) or evaluating final glycerolconcentrations that were lower than that used in the initial studies.Based on these studies, it was determined that glycerol concentrationsas low as 32.5% could be used to reproducibly maintain the viability andhistological architecture of StrataGraft® tissue during cryopreservedstorage. In contrast, a final glycerol concentration of 16.25% in thecryoprotectant solution did not support maintenance of viability infrozen tissues. See FIG. 5. By evaluating a range of glycerolconcentrations, it was determined that a cryoprotectant solutioncontaining 50% glycerol reproducibly supported cryopreservation ofStrataGraft® skin tissue and provided a margin of error above somewhatlower glycerol concentrations (e.g., 32.5%) that also supportedefficient cryopreservation.

Pre-freeze Cryoprotectant Incubation Temperature, Incubation Time, andNumber of Incubation Steps

In addition to the concentration of glycerol in the cryoprotectantsolution, three other factors affecting treatment of StrataGraft® tissuewith cryoprotectant prior to cryopreservation are: 1) the pre-freezecryoprotectant incubation time, 2) the temperature at which the tissuesare treated with cryoprotectant, and 3) the number of steps required toreach the final glycerol concentration. As described above, initialfeasibility studies with the three-step process involved sequentiallyincubating the tissues in solutions with increasing glycerolconcentrations at successively lower temperatures for 15-20 minutes ateach step. As stated above, a simpler cryopreservation process ispreferred to avoid the complexity of having multiple steps withdifferent solutions performed at different temperatures to reduce theopportunity for error and facilitate process scale-up. Toward this goal,the need for stepwise increase in cryoprotectant concentration andstepwise reduction in temperature during the pre-freeze cryoprotectantequilibration phase was evaluated.

In a series of studies performed in conjunction with evaluation ofdifferent cryoprotectant concentrations, it was determined thatStrataGraft® tissues treated with cryoprotectant solutions containing32.5, 50, or 65% glycerol in a single step at 2-8° C. for as little as15 minutes and as long as 60 minutes were all able to withstandcryopreservation with minimal loss of viability or epidermalarchitecture. Although no decline in tissue performance was observedwith cryoprotectant treatment times up to 60 minutes, relatively shortglycerol treatment times (20 to 30 minutes) were chosen in order tominimize any potential adverse effects of prolonged exposure tocryoprotectant prior to freezing.

Freezing Rate

As described above, the initial feasibility studies utilized acontrolled-rate freezer to freeze tissues at a rate of −15° C./min afterequilibration with cryoprotectant. However, the use of a controlled-ratefreezer would impose significant additional costs and is not amenable toprocess scale-up. Historically, cryopreservation of human cells has beenaccomplished at a more moderate rate of approximately −1° C./min withoutthe use of controlled-rate freezers. This is routinely accomplished byplacing vials of cells in an ultra-cold freezer in an insulated box orcontainer designed to moderate the cooling rate to approximately −1°C./min.

Process development studies were designed to determine whetherStrataGraft® tissues could be cryopreserved without using acontrolled-rate freezer. These studies demonstrated that tissues frozenby direct transfer to an ultra-cold freezer (approximately −80 C) aftertreatment with cryoprotectant performed as well as tissues frozen in acontrolled-rate freezer. See FIG. 1.

Temperature monitoring studies were performed to track the temperatureof tissues during the cryopreservation process. Following packaging inan inner sterile bag and an outer Mylar bag, StrataGraft® tissues aretransferred to a pre-cooled rack inside of an ultra-cold freezer. Eachtissue is placed in a separate slot in the freezer rack, with ample roomabove and below the tissue to allow unrestricted airflow during thefreezing process. Using temperature monitoring probes positioned withinculture dishes packaged as described above and loaded into freezer racksin this configuration, the temperature rapidly decreases toapproximately −50° C. within the first 15 minutes, further cools toapproximately −65° C. by 30 minutes and reaches a final temperature ofapproximately −80° C. after three hours. There is no significantdifference in the temperature profiles between tissues placed in thetop, middle, and bottom positions of the freezer rack. See FIG. 2.

Final Product Packaging

In initial studies, tissues were frozen in contact with a layer ofcryoprotectant solution after incubation with cryoprotectant. Althoughtissues cryopreserved in this manner exhibited good post-thawproperties, rapid thawing of tissues frozen in contact with this layerof cryoprotectant required incubation for several minutes in a 35-39° C.water bath, which would to be difficult to implement and standardize ina surgical suite. It was subsequently determined that contact with thecryoprotectant solution was not required after tissues had been treatedwith cryoprotectant. This enabled development of the final productconfiguration in which tissues are transferred to an empty sterile 100mm culture dish after treatment with cryoprotectant, where they arefrozen in contact with the bottom of the empty dish rather than beingfrozen in contact with a layer of cryoprotectant solution.

To maintain the sterility of cryopreserved tissues, the 100 mm culturedishes containing cryoprotectant-treated tissues are asepticallypackaged and heat-sealed inside of a sterile polyethylene sample bag.The inner bag is then heat-sealed inside a puncture-resistant, foodgrade, metalized polyester/polyethylene bag, which protects the packagedtissues from light, moisture, and provides a barrier to CO₂ vapor duringshipment on dry ice. The stability and comparability studies describedbelow utilized tissues packaged and cryopreserved in this configuration.

Storage Temperature

Cryopreservation of viable skin equivalents enables burn centers to haveready access to this cell-based regenerative medicine therapeutic forburns and other indications that require rapid intervention. Optimally,major burn centers would be able to maintain an inventory of the productfor use without the need to schedule a delivery on a case by case basis.In early feasibility studies, cryopreserved tissues were stored at −196°C. in a vapor-phase nitrogen freezer. Since burn centers do nottypically have liquid nitrogen storage capabilities, cryopreservationprocedures were developed that permit storage of tissue for at least sixmonths in ultra-cold freezers (−60° C. to −90° C.), which are readilyavailable in blood and tissue banks at most hospitals and traumacenters. Results of these experiments, demonstrated that while tissuesstored at −50° C. exhibited significant losses of viability over thecourse of several weeks, tissues stored at −80° C. retained levels ofviability comparable to tissues that had been stored in nitrogen vapor.See FIG. 3. These results were obtained with several independent lots ofcryopreserved tissue, confirming the reproducibility of this finding. Asdescribed in the Stability of Cryopreserved Tissues section below,analysis of tissues stored at −80° C. for six months revealed nosignificant loss of viability or changes to epidermal architectureduring storage.

Shipping Conditions

Cryopreserved StrataGraft® skin tissue will be shipped to clinical siteson dry ice for next morning delivery via commercial courier such asFedEx or UPS. The shipping container (Freezetherm FT29, Laminar Medica)is a validated dry ice shipping box that holds sufficient dry ice tomaintain the cryopreserved tissues at <−75° C. for at least 72 hours atambient temperatures of up to 35° C. to account for possible delays indelivery. Experimental data indicates that storage of cryopreservedtissues in the dry ice shipping container for >48 hours does not haveany detectable adverse effect on tissue viability or histologicalarchitecture. See FIG. 4. Following receipt at the clinical site,cryopreserved StrataGraft tissues will be stored in an ultra-coldfreezer (e.g., −60° C. to −90° C.) until use.

Pre-operative Preparation of Cryopreserved Tissue

Prior to clinical use, cryopreserved StrataGraft tissue is thawed andincubated briefly on pads saturated with culture medium to removeresidual cryoprotectant. Due to the geometry of the tissue, the thawphase is rapid. After tissues are thawed, Transwell inserts containingthe tissue are aseptically transferred to the sterile field and placedin sterile dishes containing absorbent pads saturated with culturemedium. As described below, the timing of the post-thaw incubation phaseis flexible enough to accommodate delays that could be reasonablyanticipated during clinical use.

Thaw Temperature and Time

As described in the Final product packaging section above, tissues arecryopreserved in a culture dish without a layer of cryoprotectantsolution, which allows the tissues to be thawed rapidly at ambienttemperature simply by placing the package onto a bench or table. Precisecontrol over the thaw temperature and time is not required, asexperimental data shows that tissues thawed for varying times attemperatures ranging from 22° C. to 40° C. exhibit similar post-thawproperties.

Post-thaw Incubation Solution

Buffered post-thaw incubation solutions work better than unbufferedsolutions. Tissues incubated in simple unbuffered salt solutions(lactated Ringer's or normal saline) do not survive as well as tissuesincubated in culture media-based solutions. Stratatech's SM01 culturemedia (StrataLife series) or commercially available DMEM/F12 mediabuffered with HEPES are preferred. See FIG. 6.

Post-thaw Incubation Temperature

Initial development studies found that buffered culture media were ableto support tissue viability following post-thaw incubation at both 37 Cand room temperature. However, warm post-thaw incubation temperatures(37 C) work better than cooler temps (20-25 C) for sub-optimalincubation solutions. See FIG. 6. Starting the post-thaw incubation on apad containing 37 C media that slowly cools to room temp over the courseof 15-30 minutes also works well. Higher temp seems to be most importantin the first few minutes after thaw.

Later development studies demonstrated that buffered media solutionspre-warmed to only room temperature were comparable to media warmed to37 C in their ability to support the properties of thawed tissues. Thiswas true for tissues held on either Telfa pads or Whatman pads. See FIG.8

Post-thaw Incubation Time

Tissues can be left on the media saturated pad for times ranging from 15min to 4 hr at 20-25 C or up to 2 hr at 40 C with no significant effecton tissue viability.

Stability Study

Stability of Cryopreserved Tissues

Although many of the studies described above analyzed tissues that hadbeen stored in a frozen state for only a few days or weeks, it is widelyaccepted that the majority of damage to cryopreserved cells and tissuesoccurs during the freezing and thawing stages, with relatively littleloss of cellular viability taking place during long-term storage atreduced temperatures. Long-term storage results show that cryopreservedStrataGraft® skin tissue maintains high levels of viability andhistological architecture after at least 12 months of storage atultracold temperatures. Analysis of tissues produced and cryopreservedusing the cryopreservation process described above indicates thattissues cryopreserved with this process maintain key biological,structural, and physical properties during storage for at least 12months at ultracold temperatures. See FIG. 9.

Example 3

This example describes improved cryopreservation methods for human skinequivalents utilizing a pre-freeze treatment step with cryopreservationsolutions containing 32.5% or 50% glycerol at room temperature. Theproduction process is unchanged from the current method describedpreviously. At the end of the production process, the tissues aretreated and cryopreserved as follows.

Parameter Operating Range Cryoprotectant formulation 32.5% (v/v)glycerol DMEM (1X) 10 mM HEPES (pH 7.0 to 7.4) or 50% (v/v) glycerolDMEM (1X) 10 mM HEPES (pH 7.0 to 7.4) Pre-freeze cryoprotectantincubation Room temperature temperature Pre-freeze cryoprotectant 15-45minutes incubation time Freeze method Direct transfer to −80° C. freezerStorage temperature −70 to −90° C. Shipping conditions Overnightdelivery on dry iceCryopreservation Process Description

All steps of the cryopreservation process prior to the final productpackaging step are performed aseptically inside a Class 100 biosafetycabinet in a Class 10,000 cleanroom.

-   Step 1—Dispense 20 ml of cryoprotectant solution to 100 mm culture    dishes.-   Step 2—Transfer Transwells containing StrataGraft® tissues into    individual dishes containing cryoprotectant solution. Incubate    tissues 15-45 minutes in cryoprotectant.-   Step 3—Transfer treated StrataGraft® tissues to new sterile 100 mm    culture dishes containing final product label so that the tissue    rests on the bottom of the culture dish. Excess cryoprotectant is    allowed to drain from the skin equivalent to provide a treated skin    equivalent that is substantially free of excess cryoprotectant on    the exterior surfaces of the skin equivalent.-   Step 4—Heat-seal 100 mm culture dishes in clear, sterile bags. Place    primary package into secondary Mylar bag and heat-seal.-   Step 5—Remove the packaged StrataGraft® tissues from cleanroom and    transfer tissues to an ultralow freezer (−75° C. to −80° C.). Place    tissues in a pre-cooled rack in the freezer that allows unrestricted    airflow to the top and bottom of the packaged tissues to ensure    uniform and rapid cooling. Leave tissues undisturbed overnight    during the freezing process.

Cryopreserved tissues were thawed at room temperature for 10 minutes,transferred to a hold chamber containing Telfa pads saturated with 40 mlof HEPES-buffered culture medium that had been warmed to roomtemperature, and held at RT for 15 to 20 minutes. Tissues weretransferred to a culture dish containing 90 ml of SM01 medium andreturned to culture overnight. Tissues were analyzed for viability afterovernight re-culture. Tissues pre-treated with 32.5% glycerol at roomtemperature for 15 to 45 minutes had acceptable post-thaw viability.Tissues treated with 50% glycerol at room temperature for 15 minutesalso had acceptable viability; however, tissues treated with 50%glycerol at room temperature for 45 minutes had unacceptable viability.See FIG. 10.

Example 4

MTT assays are preferably conducted as follows. Samples are excised fromthe skin tissue using an 8 mm biopsy punch. The samples are transferredto 0.3 ml MTT Assay Medium (1 mg/ml MTT reagent in Ham's F-12) in a24-well plate that has been pre-warmed to 37° C./5% CO₂. The samples areincubated for 85-95 minutes at 37° C./5% CO₂. The samples are blottedand transferred to 2 ml isopropanol. The samples are thoroughly mixed tocompletely extract the purple formazan product. 200 μl in triplicate ofeach extract is aliquoted into a 96-well plate, using isopropanol as ablank. The absorbance (550 nm) is measured in a spectrophotometer.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in tissueculture, molecular biology, biochemistry, or related fields are intendedto be within the scope of the following claims.

We claim:
 1. A method of cryopreserving an organotypically cultured skinequivalent to maintain viable tissue comprising: treating anorganotypically cultured skin equivalent in a cryoprotectant solution ina single step, wherein said organotypically cultured skin equivalentcomprises stratified squamous epithelia on a dermal layer comprisingfibroblasts embedded in collagen, and wherein the cryoprotectantsolution comprises glycerol in an amount that is about 21% to about 70%of the cryoprotectant solution by volume; separating the treatedorganotypically cultured skin equivalent from excess cryoprotectantsolution to provide a treated skin equivalent that is substantially freeof excess cryoprotectant on the exterior surfaces of the skinequivalent; packaging said organotypically cultured skin equivalent inthe absence of additional cryoprotectant to provide a packaged skinequivalent; and freezing said packaged organotypically cultured skinequivalent to provide a cryopreserved skin equivalent that retainsviability after thawing.
 2. The method of claim 1, wherein said freezingfurther comprises freezing at about −80 C.
 3. The method of claim 1,wherein said freezing further comprises direct exposure to temperaturesranging from about −50 C. to −100 C.
 4. The method of claim 1, whereinsaid packaging further comprises enclosing said cryopreserved skinequivalent in a sterile bag and enclosing said sterile bag in a secondbag.
 5. The method of claim 1, wherein said organotypically culturedskin equivalents comprise NIKS cells.
 6. The method of claim 5, whereinsaid NIKS cells comprise an exogenous nucleic acid sequence encoding anexogenous polypeptide.
 7. The method of claim 1, wherein saidcryopreserved skin equivalent has an A₅₅₀ of at least 50% of a referenceskin equivalent as determined by an MTT assay.
 8. The method of claim 1,further comprising thawing said cryopreserved skin equivalent andapplying said thawed skin equivalent to a patient in need thereof,wherein said thawed skin equivalent is not rinsed prior to saidapplication to said patient.
 9. A method of producing a cryopreservedskin equivalent comprising: providing a culture dish comprising aninsert movable between upper and lower positions in said culture dish,said insert having a bottom planar surface formed from a porousmembrane, forming a dermal equivalent comprising fibroblast cells onsaid porous membrane in said insert, wherein said insert is placed insaid upper position in said culture dish, culturing said fibroblastcells to form a dermal equivalent, applying keratinocytes to said dermalequivalent, culturing said keratinocytes in a culture medium underconditions such that said keratinocytes form a skin equivalentcomprising stratified epithelium, removing said culture medium, loweringsaid insert to said lower position, treating said skin equivalent with acryoprotectant solution wherein the cryoprotectant solution comprisesglycerol in an amount that is about 21% to about 70% of thecryoprotectant solution by volume, separating the treatedorganotypically cultured skin equivalent from excess cryoprotectantsolution to provide a treated skin equivalent that is substantially freeof excess cryoprotectant on the exterior surfaces of the skinequivalent, and freezing said skin equivalent in said culture dish inthe absence of additional cryoprotectant to provide a cryopreserved skinequivalent that retains viability after thawing.